Pregna-1,4-dienes and compositions containing same



United States Patent 3,134,718 PREGNA-1,4-DIENES AND COMPOSITIONS CONTAINING SAME Arthur Nobile, Roseland, N..l., assignor to Schering Corporation, Bloomfield, N.J., a corporation of New Jersey No Drawing. Filed Dec. 12, 1963, Ser. No. 329,959

25 Claims. (Cl. 167-65) This invention relates to an improved microbiological treatment of certain pregnenes whereby desirable chemical changes are effected without undesired degradation of the steroid molecule, such as splitting of the D-ring, or degradation of the side chain and to products produced thereby.

It is the general object of the invention to provide new compounds related to the cortical hormones and others having improved physiological activity.

It is also an object of the invention to eifect chemical modification of certain pregnene compounds by a microbiological treatment thereof or by treatment with an enzymatic extract of the microbial culture whereby they are converted into 1,4-pregnadiene compounds with or without simultaneous oxidation of a secondary hydroxyl to a keto group.

More specifically, it is an object of the invention to accomplish one or more of the operations of oxidation, dehydrogenation and hydrolysis, and including the introduction of a second double bond into the A-ring of certain steroid compounds singly unsaturated in such ring, by subjecting them to the action of a culture of a member of the family of Corynebacteriaceae preferably of the genus Corynebacterium, and particularly of the species Corynebacterium simplex (American Type Culture Collection 6946) or Corynebacterium hoagii (A.T.C.C. 7005) or of an enzymatic extract of such cultures.

A number of chemical transformations of steroids by microorganisms have recently been developed which have involved the introduction of one or more hydroxyl groups into the steroid nucleus, or oxidation of hydroxyl groups to keto groups but without afiecting the degree of saturation of the steroid nucleus. More recently there has been published the chemical transformation of progesterone by the use of microorganism, such conversions involving the introduction of a double bond in ring A, but with scission of carbon-to-carbon-linkages in the side chain as well as in ring D (Fried, Thoma and Klingsberg, Oxidation of Steroids by Microorganisms. III. Side Chain Degradation, Ring D Cleavage and Dehydrogenation in Ring A, J.A.C.S. 75, 5764 (1953)). As described in this publication, fermentation of progesterone with Streptomyces lavendulae afforded 1,4-androstadien- 3,17-dione, and also 1,4-androstadien-p-ol-3-one, known to be useful as intermediates in the synthesis of estradiol and estrone. This process thus involves the introduction of a A -double bond into progesterone, but with complete degradation of the side chain. With other microorganisms there are obtained not only elimination of the side chain, but also cleavage between carbon atoms 13 and 17. Thus, fermentation of progesterone with Penicillium chrysogenum gave the known testololactone, without introduction of a new double bond into ring A.

The above-named authors state further that Biooxida- 3,134,718 Patented May 26, 1964 second double bond into the A-ring of steroid compounds already possessing a double bond in such ring (in the 4-position), without simultaneous degradation of the side chain and without splitting of the D-ring by fermenting them in a culture of a microorganism of the family Corynebacteriaceae, or by treating them with an enzy- 'matic extract of such culture, whereby compounds of improved physiological activity, and also compounds capable of conversion by known means into physiologically active compounds, are obtained.

It is also an object of the invention to provide a simple and inexpensive process for effecting other chemical transformation in steroid compounds with the aid of the abovenamed microorganisms, both with and without the introduction of a double bond into ring A, all without splitting of the carbon skeleton of the original starting compound.

A preferred embodiment of the invention sought to be patented in one of its composition aspects resides in the concept of an anti-inflammatory steroid having a molecular structure represented by a A -pregnane having a keto group at the 3- and 20-positions, hydrogen at the 9mposition, hydroxy at the and 21-positions and being characterized by the presence of a double bond in the 1-position and a member of the group consisting of keto and fl-hydroxy at the ll-position; to 21-pharmaceutically acceptable esters thereof; and to pharmaceutical formulations containing such compounds.

A further embodiment of the invention sought to be patented in another of its composition aspects resides in the concept of a A -pregnene having a keto group at the 3- and 20-positions, hydrogen at each of the 9a and Not-positions and a member of the group consisting of hydrogen, hydroxy and pharmaceutically acceptable esters thereof at the Ill-position and being characterized by the presence of a double bond in the l-position and a member of the group consisting of (H, H), O and (H, OH) at the ll-position.

Other objects and advantages of the invention will become apparent to those skilled in the art from the following more detailed description including the pharmaceutical formulations and manner of use.

I have found that the chemical modifications of 10,13- i dimethyl steroids, and especially and most importantly the A-ring dehydrogenation of steroid compounds already singly unsaturated in the A-ring, with or without one or more of the operations of oxidation, reduction, and ester-hydrolysis, can be accomplished in an efiicient and inexpensive manner by incubating or fermenting the starting-steroid with a culture medium containing an organism of the family Corynebacteriaceae (or the enzymatic extract thereof), the nature of the chemical transformation or transformation depending upon the character of the starting compound.

In the preferred manner of carrying, out the present invention there is accordingly employed a culture (or its enzymatic extract) of a dehydrogenating member of the family Corynebacteriaceae, which includes the genera Oorynebacterium, Listeria, and Erysipelothrix. The last two of these genera include the species monocytogenes, rhusiopathiae, murisepticus and erysipeloidia, which are highly pathogenic bacteria, and their commercial use consequently introduces the problem of protecting the personnel against infection. While, therefore, the members of these two genena which act to dehydrogenate the A-tring of 10,13-dimethyl steroids can be employed in the process of this invention if proper precautions are taken, it is preferred to employ members of the genus Corynebacter-ium, as the latter includes many species which are non-pathogenic in character. Good results have been obtained with the species Corynebacterium simplex and Corynebacterium hoagii, the first of which is a soil bacterium, while the second is found in the human throat (where it apparently produces no pathologic condition) and sometimes as a contaminant of cultures exposed to the atmosphere, although its real or original habitat is not known. As best results have been obtained with Corynebacterium simplex, the invention will be further described mainly with particular reference to this organism, but it will be understood that other steroid-modifying, and particularly dehydrogenating members of the family Corynebacteriaceae can be employed in place of Corynebacterium simplex.

The starting steroid compounds can be of great variety, and I have not found any group of 10,13-dimethy1 steroids which is not transformed chemically by the above named microorganism to produce a l-dehydro compound. Thus, as disclosed more in detail hereinbelow, the culture is eifective on various pregnenes, androstenes, including 17-methyl and 17-ethyl androstenes, and on sapogenins. The presence of a free hydroxyl group appears to promote the chemical transformations, but such transformations occur even though the hydroxyl group is itself oxidized to a keto group.

The starting compounds can have hydroxyl, keto, halogen, and ester groups in various positions of the nucleus or side chain; thus, hydroxyl groups may be present in the 3, 11, 17, 20 or 2l-positions; keto groups may occupy the 3, 11, 17, 20 or Zl-(aldehydo) positions, while halogen, such as fluorine or chlorine may be attached to the 9-carbon, or at other points of the nucleus or side chain. Ester groups, including and preferably the esters of acids usually employed in steroid synthesis and in preparing steroid hormones for therapeutic use, and particularly of the lower alkanoic acids, may be located at the 3, 11, 17, 2G or 2l-positions. The hydroxyl groups at the 3, 11, and 17-positions can be either the ocor fl-epimers.

By the process of the present invention, I have been able to convert 4-pregnene-17a,21-diol-3,11,20-trione into 1,4-

od oii :93 Orgy wherein X=H =0 or (H, OH) Y=CO-CH OR (R=H or acyl) (I D .OQU

The products from these reactions possess the same pharmacodynamic properties as do the corresponding hormones, cortisone, hydrocortisone, progesterone, ethinyltestosterone, methyltestosterone, corticosterone, desoXycorticosterone and ll-dehydrocorticosterone (in all of which the A-unsaturation is absent), but to a considerably enhanced degree, which makes them valuable agents in the treatment of the many diseases and conditions for which the parent hormones are employed.

By the process of the present invention, I have been able to convert for example, 4-pregnene-17a,21-diol-3,l1,20- trione (cortisone or Compound E) into 1,4-pregnadiene- 17a,2l-diol-3,l1,20-trione (l-dehydrocortisone or A-dehydrocortisone) 4-pregnene l1B,l7a,21-triol-3,20-dione (hydrocortisone or Compound F) into 1,4-pregnadiene- 11p,17u,2.l-triol-3,20-dione (A -dehydro-hydrocortisone or l-dehydrohydrocortisone or l-dehydrocortisol) and the corresponding 3-hydroxy and 3-ester compounds, i.e., 5- pregnene-3,l7a,2l-triol-11,20-dione and 5-pregnene-3,17u, Zl-triol-ILZG-dione and 5-pregnene-3,1lfl,i7a,2l-tetrol- ZO-one and their 3-esters, into the corresponding 3-keto- 1,4-pregnadiene end products. An ester group may be present not only in the 3-position but also in one or more of the ll, 17 and 2l-positions. Where an ester group is present in the 3-position, it is hydrolyzed and the resulting hydroxyl group oxidized to a keto group in the course of the reaction. The ester groups in the 11- and l7-positions are generally not hydrolyzed, at least not to any significant extent; while an ester group in the 21- position may or may not be hydrolyzed, depending on the reaction conditions. Thus, where the starting compound is a 3,2l-diester, the reaction product may be a 3-keto-21-ester compound, or a 3-keto-2l-hydroxy compound. Along with 3-hydroxyl, also ZO-hydroxyl will be oxidized to a keto group. It will thus be seen that the organism employed in the present invention is selective with respect to the oxidation step, this being limited practically completely to the 3- and ZO-positions, while the hydrolysis may be restricted to 3-ester groups.

I have found that deacetylation at the 2l-position occurs most readily at a pH of 6.87.l, and a temperature of about 26 to 29 C. Hydrolysis is greatly diminished below a pH of 6.5 and at a temperature above 32 C. On the other hand, the introduction of the A -double bond proceeds satisfactorily outside of the pH and temperature ranges at which deacetylation proceeds most actively; that is, the dehydrogenation occurs at a satisfactory rate at a pH of 7.68.0 at a temperature of 32 to 37 C. These conditions are therefore to be employed if hydrolysis of a 2l-ester is to be minimized.

The process of the invention is applicable also to fluoro, 9a-bromo, and 9ot-Chl0l0 derivatives of cortisone and hydrocortisone, yielding the corresponding pregnadienes. General transformation effected by my improved process as applied to corticoids may be represented schematically as follows:

CI'IgOR wherein H flgffi t A m C E )3 0 h as t Ogfi (IJHQOH V I 0:0 O i 6) 0 moon H0- o=o LOH position of the fl O I I O other specific reactions effected by the process of the invention are illustrated by the following:

the dotted line indicating the alternative double bond.

Specific reactions effected by the process of the invention are illustrated by the following:

CHzOGOOCH 5 0 (\I L c l i CHzOH TABLE I.OBSERVED TRANSFORMATIONS Starting Material Products Technical Name Common Name -Androstene-3fl-ol-17-one. Dehydroepi- 4-Androstene-3J7- androsterone. dione. 4-Androstenc-17fl-ol-3-one.- Testosterone (1,4-Androstadiene-3,

17-dionc), (1,4- Androstadicne-NB- o1-3-one). 5-Pregncne-3fi,20-diol lgtd-Prcgnadieneefloione. 4-Pregnene-17a,21-diol- Reichsteins 1,4-Pregnadienc-17a,21-

3,20d1one. Compound S. diol-3,20-dione and lA-Pregnadicneqm, 206,21-triol-3-one.

(l,4-Androstadicne- 3,17-dione), (1,4- Androstadienc-ITB- 01-3-0110.)

5-Androstone-3flJ7fl-diol.

17-1\Ietl1yl-4-audrostene- Methyl 17-Wethyl-1A- 17B-ol-3one. testosterone. 23mdr0stadicne-17fi-O one. 17-Ethinyl-4-androstcnc- Ethin yl- 17Ethinyl-1,4-

17B-ol-3-one. testosterone. androstadicnc-HB-ol- 3-one. 4-Prcgnene-21-ol-3,20- Desoxyeorti- 1,4-Ircgnadiene-2l-oldiouc. costerone. 3,20-dione. Qa-Fluoro--pregnene-l 119 Fluoro Compound 9a-Fluoro-1,4-pregna- 17a,21-triol-3,20-dione. F. dienc-11fl,17a,21-

triol-3,20diono.

The importance of my invention is many-fold. It is now possible to prepare easily and directly previously unknown, as well as known, steroid hermone substances containing an additional A -unsaturation. The heretofore unknown compounds include the A -derivatives of cortisone, hydrocortisone, corticosterone, Kendalls Compound A, testosterone, methyland ethinyl-testosterone, and progesterone, to name a few.

As indicated heretofore, the potency and therapeutic applicability of a A -mono unsaturated steroid hormone, especially the corticoids, are considerably enhanced by the addition of a A -bond and in particular in those compounds exhibiting anti-inflammatory effects. For example, in the widely accepted eosinophile test (Rosenberg, E., et al., Endocrinology 54, 363 (1954) for cortical hormone activity, the dienes corresponding to cortisone and hydrocortisone are several times as potent as the corresponding natural hormones.

Furthermore, it is now possible to convert S-androstene- 3/3,20-diol directly and in one step to 1,4-androstadiene 17B-ol-3-one and 1,4-androstadiene-3,l7-dione, which are valuable intermediates in the preparation of estradiol and estrone. The process of the present invention provides a much more efiicient way of preparing these intermediates, and others than has been described heretofore.

In clinical testing, the diene derivative of hydrocortisone has been found to be considerably more effective in a daily dose of 30 to 50 mg. than 75 to 100 mg. of cortisone acetate or of hydrocortisone. Whereas a patient standardized on a 75 to 100' mg. dosage of cortisone acetate, or of hydrocortisone, would require 90 to 110 mg. of codeine per day to be relieved to pain when the dosage of the cortisone acetate or hydrocortisone was reduced to 50 mg. per day, this dosage (50 mg.) of the diene derivative of hydrocortisone required no supplemental treatment with codeine and has given complete relief from pain. In fact, a daily oral dose of 50 mg. of the diene derivative of hydrocortisone had even more favorable results than a daily oral dose of 75 to 100 mg. of cortisone acetate or 8 of hydrocortisone as it gave greater relief from pain, as evidenced by the ability of the arthritic patient to clench the fist, walk briskly, and engage in other muscular activity without pain.

Dramatic and immediate improvement has been reported in a group of patients who had suffered from rheumatoid arthritis for periods ranging from 2 /2 to 25 years on treatment with l-dehydrocortisone, the improvement being both subjective and objective. Some of these patients had received no steroid therapy, while others had received, besides aspirin, also cortisone, hydrocortisone, corticotropin, g ld compounds, phenylbutazone, or terramycin, but in each case the results had been unsatisfactory. The appearance of objectionable side effects had discouraged increase in the dose of cortisone. l-dehydrocortisone had been administered orally every 8 hours, three times daily. The initial dosage ranged from 30 to 60 mg, while the maintenance doses ranged from 5 to 25 mg. daily, no other drug being administered during the course of treatment. In all cases, easing of joint pain and diminution of stiffness accompanied by a distinct feeling of well being was noted by the patients within 4 to 6 hours after the first dose of 10 mg. was taken. In most patients, increased temperature and redness of periarticular tissues disappeared at the end of 24 hours. Swelling, tenderness and pain on motion were markedly reduced and the range of motion increased in some patients at the end of only 24 hours, and the improvement progressed as the treatment continued.

The anti-inflammatory properties of particular steroid compounds of the instant invention, especially 3,20-diket0- 17ot,2l-dihydroxy-4-pregnenes (or 21-esters thereof) having an ll-keto or hydroxy group and characterized by an additional double bond in the 1-position have been found to be of great value therapeutically. These may be administered in suflicient amount to cause complete suppression of rheumatoid arthritis with marked absence or diminution of side effects such as retention of sodium, loss of potassium or nitrogen, and gain in body weight.

Maintenance doses of 5 to 25 mg. of l-dehydrocortisone and 1-dehydro-hydrocortisone, as determined by clinical response, are at least equal in their objective and subjective eifects to from 50 to 112.5 mg. of cortisone. These relatively small maintenance doses of l-dehydrocortisone have produced prompt improvement in patients who had failed to respond to larger doses of cortisone and hydrocortisone. The side eifects with my new preparation are also much less severe and less common than have been observed in cortisone therapy, indicating a higher therapeutic ratio than that of cortisone.

In order to obtain a desirable growth of Corynebacterium simplex (American Type Culture Collection 6946) for the process of this invention, a suitable nutrient medium is prepared containing carbohydrate, organic nitrogen, cofactors, and inorganic salts. It is possible to omit the use of carbohydrate without completely impairing the growth of the organism. The steroid compound as a solid or dissolved or suspended in ethanol, acetone or any other water-miscible solvent which is non-toxic toward the organisms, is added to the cultivated microorganism in a broth medium under sterile conditions. This culture is then shaken, aerated, or simultaneously aerated and agitated, in order to enhance the growth of the Corynebacterium simplex and the biochemical conversion of the steroid substrate. The steroid may be added to the broth medium and then inoculated with the bacterium, or the cultivated microorganism in broth medium may be added to the steroid. In certain cases, depending on the conditions of the reaction medium, it may be more desirable to obtain optimum growth of the microorganism before the addition of the steroid. Alternatively, enzyme preparations obtained in known manner from cultures of Coryncbacterium simplex may be used for carrying out the process.

A useful method for carrying out the process is the cultivation of Corynebacterz'um simplex on a suitable nutrient medium under aerobic conditions. After cultivation of the microorganism, the cell mass may be harvested by centrifuging the nutrient broth, decanting the supernatant liquid and suspending the cell mass in saline. A suitable volume of the cell suspension is then seeded into a desirable nutrient medium for supporting growth of the microorganism. The nutrient medium employed may be a yeast extract (Difco) casein hydrolysate (N-Z- Amine) (Type B Sheflield), corn steep liquor, water extract of soybean oil meal, lactalbumin hydrolysate (Edamine-Sheffield Enzymatic), fish solubles, and the like.

Inorganic salts are desirable to maintain a pH level in the reaction medium of between 6.8 and 7.2. However, the use of inorganic salts for buffering the reaction mixture may be omitted. The omission of inorganic salts causes the pH to rise from an initial value of 6.8 to about 7.743. This, however, will still permit the formation of the desired steroidal and products. The optimum temperature for growth of the selected microorganism is 37 C., but the temperatures may vary between 25 and 37, and even between 20 and 40 C. The time of reaction may vary from as little as 3 hours to as much as 48 hours. The length of time which is employed will depend on the steroid which is being transformed. Any water miscible, non-toxic (to the organism) solvent may be employed to dissolve or suspend the steroid. I prefer to use ethanol or acetone in such amounts that the final concentration of these solvents in the reaction mixture is no higher than about 7% and may amount to only traces; owing to evaporation, the final concetration of the organic solvent may even be practically zero.

Following the completion of the oxidation or dehydrogenation process, which may be accompanied by partial or complete hydrolysis when monoor poly-esters are used, the products of reaction may be recovered from the mixture by extraction with a suitable water-immiscible solvent, by filtration, by adsorption on a suitable adsorbent, or by any of the other procedures commonly used in the art. For extraction, chlorinated lower hydrocarbons, ketones, and alcohols are useful. These include chloroform, methylene chloride, trichloroethane, ethylene dichloride, butanol, diethylketone, and others. I prefer to use extraction as the method for isolating the steroidal products. Following extraction, the products may be isolated by concentration of the extracts to a small volume or to dryness. Purification of the residues may be then accomplished in several Ways. In many instances, as with the diones of cortisone and hydrocortisone, simple recrystallizations from a suitable solvent or solvent mixture, such as acetone, methylene chloride, ethanol, acetonehexane, methylene chloride-hexane, etc. affords the desired dienone in excellent yield and high state of purity. Where there are several products formed in the same reaction, a separation is conveniently achieved by chromatography on silica gel, magnesium silicate (Florisil), alumina or other commonly employed adsorbents. It should be understood that where a mixture of more polar steroids is to be separated, more polar eluents will be required in chromatographic separation. Furthermore, the activity of the adsorbent varies from batch to batch and consequently the same steroids may be more strongly or more weakly hold in parallel experiments which will result in variation of the value of the concentration of the eluents which will elute a given steroid. In any case, the

order of elution will remain the same for a given adsorbent and a given solvent system.

The 1,4-diene structure of the products has been established in various ways. The steroids described herein are new substances and careful attention has been paid to the proof of their structures. Infrared analyses, ultraviolet analyses, rotational analyses, degradative studies, and carbon-hydrogen analyses have been employed where necessary to establish the nature of the products. The introduction of" the A unsaturation in a 3-keto-A steroid is known to have a characteristic negative effect on the molecular rotation of the product, and I have observed this shift in rotation in the dienones which I have prepared from cortisone and hydrocortisone among others.

The chemical transformations which can be accomplished by subjecting the various 10,13-dimethyl steroids to the action of a culture of Corynebacterium simplex (the term culture of Corynebacterium simplex is to be understood in this specification and in the claims as including the enzymatic extract of such culture) are thus of widely difierent kinds, and can take place singly, or two or more of such transformations can occur simultaneously or in sequence. The various reactions appear to be unaifected by other substituents in the steroid nucleus or in the side chain.

The reactions of the present invention include the conversion of 5-pregnene-3,17a,20,21-tetrol-1l-one and its 3- and/ or 21-esters into 1,4-pregnadiene-l7a,2l-diol-3,11,20- trione and its 2l-ester; and of 5-pregnene-3,lla,17a,20,21- pentol and its 3- and/or 21-esters into 1,4-pregnediene- 1la,l7a,2l triol-3,20-dione and its 2l-ester.

While the lower alkanoic esters are generally preferred, and particularly the acetates, as above indicated, it will be understood that the specific character of the ester is not controlling in my process and that other esters, both of organic and inorganic acids may be employed, such as cyclopentyl and cyclohexyl acetates, propionates and butyrates, and also the phosphates, polyphosphates and sulfates, it being necessary only that the esters be nontoxic toward the microorganism. The hydroxylated products, especially the 2l-hydroxylated anti-inflammatory dienes can, if desired, be converted into their coresponding Zl-esters, by known procedures, such as later de scribed herein.

The therapeutically active dienes of the present invention are preferably administered by mouth in the form of tablets containing, for example, about 5 to 20 mg. per tablet mixed with a solid carrier containing one or more of the usual ingredients, such as starch, sugar, gums, scaps, clays and the like. They may, however, be also administered by subcutaneous or intramuscular injection, dissolved, or suspended in a suitable non-toxic liquid vehicle; or they canbe administered in the solid form by subcutaneous implantation, or in the form of suppositories dissolved or suspended in a fatty or waxy vehicle which melts at approximately body temperature. They can also be adminsitered topically in the form of an ointment or cream in which they are dissolved or suspended in an unguent or cream base of known composition; and they may also be employed in the form of ointments and aqueous suspensions for ophthalmic use. The compounds in microcrystalline form in aqueous suspensions can be used for intra-articular injection and also as nasal spray; while infusions can be prepared for intravenous use.

The fish solubles referred to hereinabove are presently available commercially as an extract of herring, menhaden, and various mixtures thereof, which has been subjected to an enzymatic hydrolysis. This material can be added directly to the culture broth for supplying nutrient material. Where fish solubles (50% solid content) are available which have not been subjected ot enzymatic hydrolysis, such extracts should be diluted with water and steamed for about 10 minutes to C. followed by filtration, preferably with the aid of diatomaceous earth.

This application is a continuation in-part of my copending applications, Serial No. 449,257, filed August 11, 1954; and Serial No. 492,773 filed March 7, 1955, both now abandoned.

The invention will be-described in further detail in the following examples which are presented by way of illustration only and not as indicating the scope of the invention.

1 1 EXAMPLE 1 Conversion of Cortisone to 1,4-Pregnadiene- 1 7a,21-Di0l-3,11 ,ZO-Trione From a solution of 30 g. of yeast extract (Difco) in 3.0 l. of tap water containing 13.2 g. of potassium dihydrogen phosphate and 26.4 g. of disodium hydrogen phosphate (pH of the solution 6.9), 27 portions of 100 ml. each are withdrawn, placed in 300 ml. Erlenmeyer flasks and sterilized by autoclaving for 15 minutes at 15 lb. steam pressure (120 C.). After autoclaving and cooling of the broth one ml. of a suspension of Corynebacterium simplex (A.T.C.C. 6946) is placed in each flask. The flasks are then shaken on a shake table at 220 r.p.m. and 28 C. for 24 hours.

Into each of 27 Erlenmeyer flasks are placed 150 mg. of cortisone. The flasks and contents are then sterilized for 15 minutes at 15 lb. steam pressure (120 C.). To each flask are then added 5.0 ml. of ethanol. The 24- hour bacterial culture is then transferred aseptically and the resulting suspensions are shaken on a shake table at 220 r.p.m., and 28 C. for 48 hours. The final pH is 7.2.

The contents of all the flasks are combined and extracted with a total of 9.0 l. of chloroform in three equal portions. The combined extracts are then concentrated to a residue which is crystallized from acetone-hexane. There results 1.1 g. of l,4-pregnadiene-17a,2l-diol-3,11, 20-trione, M.P. 210215 C. (dec.). Several additional recrystallizations raised the M.P. to 230232 C. (dec.); +175.3 (dioxane); 6 15,400 (methanol).

Analysis.--Calcd. for C H O C, 70.37; H, 7.31. Found: C, 70.38; H, 7.67.

The infrared spectrum of the product shows the presence of a 1,4-diene-3-one system, hydroxyl and additional carbonyl (6-membered ring or side-chain). The structure of the product is established as 1,4-pregnadiene-17u,21- diol-3,11,20-trione by degradation to 1,4-andrstadiene-3, 11,17-trione (identical with an authentic sample) and by formation of a monoacetate by the action of acetic anhydride-pyridine which shows a characteristic interaction of the C-20 carbonyl with the C-21 acetate in the infrared spectrum.

21-A cetylation of 1,4-Pregnadz'ene-17oc,21-

Dial-3,11,20-Tri0ne To a solution of 0.5 g. of 1,4-pregnadiene-l7u,2l-diol- 3,11,20-trione in ml. of anhydrous pyridine are added 3 ml. of acetic anhydride. The reaction mixture is permitted to stand overnight at room temperature, and is then diluted with ice and water. The resulting precipitate is filtered and recrystallized from acetone-hexane. There is obtained 0.35 g. of 1,4-pregnadiene-17a,2l-diol- 3,11,20-trione 21-acetate, M.P. 227228 C. (dec.). After several recrystallizations from acetone-hexane it melted at 233236 C. (dec.).

EXAMPLE 2 Conversion of Hydrocortisone to 1,4-Pregnadienel15170521-Tri0l-3,20-Di0ne From a solution of 3 g. of yeast extract (Difco) in 3.0 l. of tap water containing 13.2 g. of potassium dihydrogen phosphate and 26.4 g. of disodium hydrogen phosphate (pH of the solution, 6.9) 27 portions of 100 ml. each are withdrawn, placed in 300 ml. Erlenmeyer flasks and sterilized by autoclaving for 15 minutes at 15 lb. steam pressure (120 C.). After autoclaving and cooling of the broth, one ml. of suspension of Corynebacterium simplex (A.T.C.C. 6946) is placed in each flask. The flasks are then shaken on a shake table at 220 r.p.m. and 28 C. for 24 hours.

Into each of 27 Erlenmeyer flasks are placed 150 mg. of hydrocortisone. The flasks and contents are then sterilized for 15 minutes at 15 lb. steam pressure (120 C.). To each flasks are then added 5.0 ml. of ethanol. The 24-hour bacterial culture is then transferred aseptically 12 and the resulting suspensions are shaken on a shake table at 220 r.p.m. and 28 C. for 48 hours. The pH at the end of the shake period is 7.0.

The contents of all the flasks are combined and extracted with a total of 9.0 1. of chloroform in three equal portions. The combined extracts are then concentrated to a residue which weigh 3.75 g. The M.P. of the residue is 227-232 C. From 2.75 g. of this crude material on sludging with 50 ml. of acetone and cooling, there is recovered in filtration 1.35 g. of 1,4-pregnadiene-1 1 53,1711, 21-triol-3,20-dione, M.P. 237239 C. (dec.). Additional product can be recovered from the mother liquor. Recrystallization from acetone raised the M.P. to 239241 C. (dec.); +107 (dioxane): e =14,600 (methanol).

Analysis.Calcd. for C H O C, 69.97; H, 7.83. Found: C, 70.24; H, 8.13.

The infrared spectrum indicates the presence of a 1,4- diene-3-one system, hydroxyl and a 6-membcred ring or side chain ketone (in addition to the dienone carbonyl). From this evidence and the fact that a mono-acetate is formed with acetic anhydride-pyridine whose infrared spectrum shows the expected interaction between the C-21 acetate and the C-20 carbonyl group, the product of fermentation is proven to possess the assigned structure.

21-Acetylation of 1,4-Pregnadiene-1 1 8,] 701,21- Trial-3,20-Dione To a solution of 0.85 g. of 1,4-pregnadiene-115,111,21- triol-3,20-dione in 5 ml. of pyridine are added 3 ml. of acetic anhydride. The reaction mixture is allowed to stand at room temperature overnight and is then diluted with ice water. The resulting precipitate is filtered from the mixture and recrystallized from acetone-hexane. There is recovered 0.45 g. of 1,4-pregnadiene-l1,8,17a,21- triol-3,20-dione 2l-acetate, M.P. 235239 C. On recrystallization, the M.P. rose to 237239; [01] +116 (dioxane): 5 15,000 (methanol).

Analysis.-Calcd. for C H O C, 68.63; H, 7.51. Found: C, 68.62; H, 7.78.

EXAMPLE 3 Conversion of Hydrocortisone ZI-Acetate to the 1,4-Diene and its ZI-Acetate The reaction is run exactly as described for the transformation of hydrocortisone to the corresponding diene (Example 2), and the product is isolated by chloroform extraction and crystallization from acetone. From 1.0 g. of hydrocortisone 2l-acetate there is obtained 0.22 g. of 1,4-pregnadiene-l1e,17a,21-triol-3,20-dione, M.P. 239- 241 C. (dec.).

When it is desired to suppress the deacetylation reaction, the same conditions as above are used with the exception that the temperature of the environment for the growth and reaction phases of the process is raised to 36 C. The product is isolated in the usual way. From 1.0 g. of hydrocortisone 21-acetate there results 0.13 g. of hydrocortisone diene-ZI-acetate, M.P. 237239 C. (dec.).

EXAMPLE 4 Conversion of Cortisone 21 -Acetate to the 1,4- Diene and its ZJ-Acetate The reaction is conducted as described for the transformation of cortisone to the corresponding diene (Example 1), and the product is isolated by chloroform extraction and crystallized from acetone-hexane. From 1.0 g. of cortisone acetate there is isolated 0.17 g. of 1,4-pregnadiene :,21 diol 3,11,20 trione, M.P. 230-232 C.

When it is desired to suppress the deacetylation, the same conditions as above are used with the exception that the temperature of the environment for the growth and reaction phases of the process is raised to 36 C. The product is isolated in the usual way. From 1.0 g. of

cortisone acetate there results 0.11 g. of cortisone diene- 21-acetate, M.P. 230-233 C. (dec.).

EXAMPLE Conversion of Compound S to 1,4-Pregnadiene-17a,21- Dial-3,20-Dione, and 1,4-Pregnadiene-17a,20,21-Triol- 3-0ne One hundred ml. of a 1.0% yeast extract concentrate including 9.0 ml. of 0.2 M KH PO and 9.0 ml. of 0.2 M Na HPO is sterilized as before and inoculated with a 1.0% suspension of Corynebacterium simplex (A.T.C.C. 6946) from a 24-hour broth culture. The newly seeded culture is incubated and shaken (shake table) for 20 hours at 28 C. After incubation the broth culture is transferred aseptically to a second sterile 300 ml. Erlenmeyer flask containing 150.0 mg. of sterile Compound S (4- pregnen-17et,2l-diol-3,20-dione) in 5.0 ml. ethanol or acetone. The pH of the reaction mixture is 7.0. The bacterial culture containing steroid and solvent is incubated and shaken for a period of 48 hours at 28 C. The final pH of the reaction mixture is 7.2-7.4. The culture is then extracted thoroughly with chloroform. The extracts are pooled and concentrated on a steam bath to dryness. The crude extract weighs 196.0 mg.

The total crude extract is sludged with methanol and there is obtained 80 mg. of crystalline solid, M.P. 246- 250 C. After two crystallizations from acetone the M.P. is 246-249 C. (dec.), +76 (CHCl E 15,500 (C H OH).

Calcd for C H O C, 73.22; H, 8.19. Found: C, 73.56; H, 8.40.

The infrared spectrum indicates the presence of the 1,4- diene-3-one system, hydroxyl and another carbonyl (6- membered ring or side-chain). The product is therefore 1,4-pregnadiene-17a,21-diol-3,20-dione.

The structure is proved by degradation with sodium bismuthate in aqueous acetic acid to 1,4-androstadiene-3,l7- dione and by acetylation to a 21-monoacetate (interaction between 2l-acetate and 20-carbonyl apparent in the infrared spectrum).

21-Acetylatz'on of 1,4-Pregnadiene-17a,21-Di0l- 3,20-Dione To a solution of 0.25 g. of 1,4-pregnadiene-17u,2l-diol- 3,20-dione in 2 ml. of pyridine was added 1 ml. of acetic anhydride. The reaction mixture was allowed to stand at room temperature overnight and was then diluted with ice and water. The resulting precipitate was filtered and recrystallized from methylene chloride-hexane, affording 0.20 g. of 1,4-pregnadiene-l7a,2l-diol-3,20-dione 21-acetate, M.P. 226.5-228 C.

EXAMPLE 6 Conversion of Compound S to 1,4-Pregnadiene-17 ,21- Dial-3,20-Dione, and 1,4-Pregnadiene-17a,20,21-Triol- 3-0ne One hundred ml. of a medium consisting of 1% fish solubles (prepared as described below), 0.1% yeast extract, 9.0 ml. of 0.2 M KH PO and 9.0 ml. of 0.2 M Na HPO are sterilized as described hereinabove and inoculated with a suspension of Corynebacterium simplex from a 24-hour broth culture. The newly seeded culture is incubated and shaken (shake table) for 20 hours at 28 C. After incubation, the broth culture is transferred aseptically to a second sterile 300 m1. Erlenmeyer flask containing 150 mg. of sterile Compound S in 5.0 ml. of ethanol. The pH of the reaction mixture is 6.9. The bacterial culture containing steriod and solvent is incubated and shaken for 48 hours at 28 C. The final pH is 7.3. The culture is extracted with 2 l. of chloroform in 5 equal portions, the extracts are pooled, and the pool is concentrated on the steam bath.

The crude residue is taken up in methylene chloride and chromatographed over Florisil. There is isolated from 14 the chromatogram, starting material (15 mg.), 1,4-pregnadiene-17a,21-diol-3,20-dione (30 mg.) and 1,4-pregnadiene-17 u,20,21-triol-3-one mg.). The previously unidentified triol is recrystallized from acetone-hexane and melts at l-190 C., [a] +33 (methanol).

Analysis.Calcd. for C H O C, 72.80; H, 8.73. Found: C, 72.79; H, 9.08.

The structure of the triol is proved by acetylation with acetic anhydride-pyridine to a 20,21-diacetate and degradation to 1,4-andros'tadiene-3,17dione. The infrared spectrum of the triol shows a 1,4-diene-3-one band, strong hydroxyl band and the absence of anything corresponding to a 20-carbonyl band. 1

In place of ethanol there can be employed other watersoluble organic solvents which are non-toxic to the microorganism, such as acetone, mixtures of ethanol and acetone, and the like.

EXAMPLE 7 Conversion of H yarocortisone to 1,4-Pregnadiene- 115,17a,21-Triol-3,20-Dione From a solution of 1 gram yeast extract (Difco) in 1.0 liter of tap water containing 4.4 gm. of potassium dihydrogen phosphate and 8.8 g. of disodium hydrogen phosphate (pH of the solution 6.9), 10 portions of ml. each are withdrawn, placed in 300 ml. Erlenmeyer flasks and sterilized by autoclaving for 15 minutes at 15 lbs. steam pressure C.). After autoclaving and cooling of the broth, one ml. of suspension of Corynebacterium hoagii (American Type Culture Collection 7005) is placed in each flask. The flasks are then shaken on a shake table at 220 r.p.m. and 28 C. for 16 /2 hours.

Into each of the 10 Erlenmeyer flasks, 50 mg. of hydrocortisone dissolved in 1 m1. of 90% methanol is added aseptically. The flasks are replaced on the shaker and incubated for 7 hours. The pH at the end of the shake period is 6.82.

The contents of all flasks are combined and extracted with a total of 3 liters of chloroform in 3 equal portions. The combined extracts are concentrated to a residue of 425 mg. Crystallization of the residue from acetone affords 248 mg. of 1,4-pregnadiene-l1B,17a,21-triol-3,20- dione.

EXAMPLE 8 Conversion of H ydrocortisone to 1,4-Pregnadiene- 11,8,17oc,21-Triol-3,20-Dione From a solution of 0.5 gram Basamin Busch (Anheuser-Busch) in 1 liter of tap water, 10 portions of 100 ml. each are withdrawn, placed in 300 ml. Erlenmeyer flasks and sterilized by autoclaving for 15 minutes at 15 lb. steam pressure (120 C.). After autoclaving and cooling of the broth, one ml. of a suspension of Corynebacterium simplex is placed in each flask. The flasks are then shaken on a shake table at 220 r.p.m. and at 28 C. for 24 hours.

Into each of the 10 Erlenmeyer flasks 50 mg. of hydrocortisone dissolved in 0.8 ml. of absolute methanol, are added aseptically. The flasks are replaced on the shaker and incubated for an additional 47 hours. The pH at the end of the shake period is 7.2-7.6.

The contents of all flasks are combined and extracted with a total of 3 liters of chloroform in 3 equal portions. The combined extracts are concentrated to a residue of 490 mg. Crystallization of the residue from acetone affords 403 mg. of 1,4-pregnadiene-l lfi,l7u,2l-triol-3,20- dione, M.P. 238-240 c. (dec.): 1 +10s (dioxane), 6 14,500 (methanol).

EXAMPLE 9 Conversion of Corlisone to 1,4-Pregnadiene-17a,21-Diol- Dial-3,11 ,ZO-Trione The procedure described in Example 8 is followed except that cortisone is used in place of hydrocortisone,

EXAMPLE 10 The 9 Conversion of 9a-Fluor0-4-Pregnene-11,B,1 7tt,Z1-Tri0l- 3,20-Dine to the 1,4-Pregnadiene A one hundred ml. broth culture containing a 0.1% acid extract concentration, 9.0 ml. of 0.2 M KH PO and 9.0 ml. of 0.2 M Na HPO contained in a 300 ml. Erlenmeyer flask is seeded with 1 ml. of a 24-hour broth culture of Coryncbacterium simplex. The flask is incubated at 28 C. for 24 hours. A second 300 ml. Erlenmeyer flask containing 100 mg. of sterile 9a-fluoro-4- pregnene-l1(3,17u,21-triol-3,20-dione in 5.0 ml. of acetone is incubated with the 24-hour culture of the bacterium. The culture containing steroid solution is incubated for 48 hours at 28 -30 C.

The culture is then extracted with three volumes of chloroform, the extracts combined and concentrated to dryness on a steam bath. Recrystallization of the residue gives 9a fluoro 1,4-pregnadiene-11 ,B,17oc-21t1i0l-3,20- dione as a crystalline solid (from acetone), M.P. 247- 250 C.

EXAMPLE 11 Reaction of -Pregnen-3B,20-Di0l One hundred ml. of a 0.1% yeast extract concentrate including 9.0 ml. of 0.2 M KH PO and 9.0 ml. of 0.2 M Na HPO is autoclaved in a 300 ml. Erlenmeyer flask. After autoclaving for minutes at 15 lbs. (120 C.), the flask is allowed to cool to room temperature. The flask is then seeded with a suspension of Corynebacterium simplex (A.T.C.C. 6946). The seeded flask is incubated and shaken (shake table) for 24 hours at 28 C.

A second 300 ml. Erlenmeyer flask containing 150 mg. of 5-pregnene-3/3,20-diol is sterilized in an autoclave for 15 minutes at 15 lbs. (120 C.). To this flask is then added 5.0 m1. of acetone or ethanol to dissolve the steroid. The 24-hour growth culture of Corynebacterium simplex is transferred asptically to the flask containing the steroid and the reaction mixture is shaken (shake table) for 36 hours at 28 C. At the end of the transformation period the pH is 7.1-7.2.

The reaction mixture is then extracted thoroughly with chloroform, the chloroform extracts are pooled and the resulting solution is concentrated to a residue (0.20 g.). The crude extract is crystallized from ether as long prisms, M.P. 135-138 C. Two crystallizations from methylene chloride-hexane afford 0.06 g. of 1,4-pregnadiene-3,20- dione, M.P. 152-153 C., +122 C. (CI-ICl 6 15,000 (C H OH). The infrared spectrum indicated the presence of a 1,4-diene-3-one system, another carbonyl (6-membered ring or side-chain), and the complete absence of hydroxyl.

EXAMPLE 12 Reaction of Dehydroisoandrosterone One hundred ml. of a 24-hour broth culture of Corynebacterium simplex (A.T.C.C. 6946) grown in a 0.1% yeast extract (Difco) and 9.0 ml. of a 0.2 M KH PO and 9.0 ml. of a 0.2 M Na HPO are transferred to a 300 m1. of Erlenmeyer flask containing 150 mg. of dehydroisoandrosterone in 5.0 ml. ethanol. The flask containing the steroid and bacterial culture is placed on a shaking machine and allowed to shake for a period of 24 hours at a temperature of 28 C. to 30 C.

From an initial pH of 6.8, in 48 hours, the reaction medium has a final pH of 7.1 to 7.2.

After the transformation period, the 100 ml. broth culture is extracted with 3 equal volumes of chloroform. The chloroform volumes are combined and concentrated 16 to dryness on a steam bath. The solid crude residue weighs 193.0 mg.

The total crude residue from the extraction of the broth is sludged with methanol, whereupon there result 30 mg. of elongated prisms, M.P. 155-165 C. The infrared spectrum of this compound is identical with that of 4- androstene-3,17-dione.

From the mother liquors of the sludge on further concentration and crystallization from ether-hexane there is obtained 70 mg. of solid M.P. 143 C. Recrystallization from ether gives 53 mg., M.P. 147-152 C. The infrared spectrum of this material is identical with that of 4-androstene-3,17-dione.

The products from the two crystallizations are combined and crystallized from ether. There result 70 mg. of 4-androstene-3,l7-dione, M.P. 167-169 C.; there is no depression of melting point on admixture with an authentic sample.

EXAMPLE 13 Reaction of 5-A ndr0sten-3{3,1 7B-Di0l One hundred ml. of a 20- hour broth culture of Corynebacterium simplex (A.T.C.C. 6946) containing a 0.1% yeast extract, 9.0 ml. of 0.2 M KH PO and 9.0 ml. of 0.2 M Na I-IPO are transferred to a 300 ml. Erlenmeyer flask containing 150 mg. of androstendiol in 5.0 ml. ethanol. The flask is placed on a shaking machine and incubated at 28 C. and is allowed to run for a period of 48 hours, the final pH of this reaction mixture being 7.1.

The broth culture is extracted with three equal volumes of CHCl The solvent volumes are combined and concentrated to dryness on a stream bath. The solid crude extract Weighs 158.0 mg.

The total crude extract is crystallized from other and there result 40 mg. of solid, M.P. 128-135 C., [M +69 (CHCla), Whose infrared spectrum contains bands characteristic of a 1,4-diene-3-one system (strong), hydroxyl (strong) and five-membered ring carbonyl (moderate). Repeated crystallization from ether-hexane and methylene chloride-hexane afford ultimately 11 mg. of 1,4- androstadiene-17p-ol-3-one, M.P. 164-l66 C., +25 (CHCl The infrared spectrum of this sample is identical with that of an authentic sample.

The mother liquor from the original ether crystallization is chromatographed over Florisil which had been prepared with hexane. Material eluted from the column with 1020% ether in hexane contains only traces of hydroxyl in the infrared spectrum and shows a strong fivemembered ring carbonyl together with the 1,4-diene-3-one bands. Crystallization of the pooled eluates of this group from ether-hexane affords 25 mg. of 1,4-androstadiene- 3,17-dione, M.P. 139-140, [a] +110 (CHCl The infrared spectrum of this material is identical with that ofan authentic sample.

EXAMPLE 14 Reaction of Testosterone A one hundred ml. broth culture containing a 0.1% yeast extract concentration, 9.0 ml. of 0.2 M KH PO and 9.0 ml. of 0.2 M Na HPO is seeded with 1 ml. of a 1.0% inoculum from a 24-hour broth culture of Corynebacterium simplex (A.T.C.C. 6946). The flask is incubated at 28 C. for 24 hours. A second 300 ml. Erlenmeyer flask containing 150 mg. of sterile testosterone in 5.0 ml. acetone is inoculated with the 24-hour culture of Corynebacterium simplex (A.T.C.C. 6946). The culturecontaining steroid solution is incubated for 48 hours at 28 C. to 30 C.

After termination of the transformation period, the pH is 7.2-7.3. The culture is now directly extracted with 3 equal volumes of CHCl the solvent volumes combined and concentrated to dryness on a steam bath. The crude extract weighs 153.0 mg.

The total crude is chromatographed on Florisil prepared with hexane. The fractions collected with 10-20% ether-hexane eluate are pooled and crystallized from ether-hexane, afi'ording 12 mg. of a solid, M.P. 134-138 C., which is shown to be identical with 1,4-androstadiene- 3.17-dione by comparison of the infrared spectra and absence of depression of melting point on admixture with an authentic sample. The fractions collected with 30- 50% ether-hexane eluate are pooled and crystallized from ether-hexane, affording 32 mg. of a solid, M.P. 159-165 C., which is shown to be identical with 1,4-androstadiene- 17fl-ol-3-one by comparison of the infrared spectra and absence of depression of melting point on admixture with an authentic sample.

EXAMPLE 15 Reaction of Diosgenin To a 300 ml. Erlenmeyer flask, containing 9.0 ml. each of 0.2 M KH PO and 0.2 M Na HPO in 1.0% yeast extract (Difco) is added in inoculum in the form of 1 m1. of a bacterial suspension of Corynebacteriaum simplex (A.T.C.C. 6946). The bacterial culture is placed on a shaking machine and incubated at 28 C. for 24 hours.

After 24 hours of incubation, the culture is transferred aseptically to a second flask containing 150 mg. of diosgenin (22u,25D-spirost-5-ene-3,B-ol) in 5.0 ml. of ethanol. The flask is then shaken for a period of from 24-36 hours.

At the end of the transformation period, the flask is extracted with 3 equal volumes of CHCl The CHCl volumes are combined and concentrated to dryness on a steam bath. The crude extract weighs 175.0 mg.

The total crude extract is crystallized from methanol and there is isolated a product whose infrared spectrum shows the presence of 1,4-diene-3-one system, a 3-keto-A system and the spiroketal side-chain. The products are chromatographed over Florisil and there are isolated diosgenome and l,4-diosgedienone-(22a-25D-spirosta 1,4 diene-3-one).

EXAMPLE 16 Reaction of 5-Pregnene-3BJ7a,21Tri0l-20-One 3,21 -Diacetate The reaction medium and organism are prepared as described in Example 14; 150 mg. of 5-pregnene-3,8,17a,21- triol-ZO-one 3,21 diacetate are added, and the reaction is permitted to proceed as previously described.

The products are extracted with chloroform, the chloroform extracts are concentrated to a small volume and chromatographed on Florisil. The order of elution is unreacted starting material (75 mg.) first, then compound S 21-acetate (15 mg.) and finally l,4-pregnadiene-17a,21- diol-3,20-dione (30 mg). The products were all identified by comparison of their infrared spectra with those of authentic samples.

EXAMPLE 17 Reaction of 4-Pregnene-3fl-0l-20-One The reaction medium and organism are prepared as described in Example 14; 150 mg. of 4-pregnene-3fl-ol-20- one are added, and the reaction is permitted to proceed as previously described. The product is extracted with chloroform and isolated by evaporation to dryness. Recrystallization of the residue from etherhexane airords 73 mg. of 1,4-pregnadiene-3,20-dione, M.P. 150-152 C.

EXAMPLE 18 Reaction of 17-Ethinyltest0slerone To the reaction of medium and organism prepared as described in Example 14, there are added 150 mg. of 17- ethinyltestosterone and the reaction is permitted to proceed as previously described.

The product is extracted with chloroform and isolated by evaporation to dryness. Recrystallization of the residue yields 17-ethinyl-1,4-androstadiene-17,6-01-3-one as a crystalline solid.

1 & EXAMPLE 19 Reaction of 17-]ifezhyltest0sterone The reaction of medium and organism are prepared as described in Example 14; mg. of l7-methyltestosterone are added and the reaction is permitted to proceed as above described.

The product is extracted with chloroform and isolated by evaporation to dryness. Recrystallization of the residue gives crystalline 17a-met-hyl-1,4-androstadiene-17fl-ol- 3-one.

EXAMPLE 20 Reaction of Corticosterone The reaction medium and organism are prepared as described in Example 14; 150 mg. of 4-pregnene-11p,21- diol-3,20-dione are added and the reaction is permitted to proceed as above described.

The product is extracted with chloroform and isolated by evaporation to dryness. Recrystallization of the residue affords crystalline 1,4-pregnadiene-11,3,21-diol-3,20- dione.

EXAMPLE 21 Reaction of Desoxycorticosterone The'reaction medium and organism are prepared as described in Example 14; 150 mg. of 4-pregnene-2l-ol- 3,20-dione are added and the reaction is permitted to proceed as above described.

The product is extracted with chloroform and isolated by evaporation to dryness. Recrystallization of the residue affords 1,4-pregnadiene-21-ol-3,20-dione as a crystalline solid.

EXAMPLE 22 Reaction of 11 -Dehydr0cortic0ster0ne To the reaction medium and organism prepared as described in Example 14, 150 mg. of 4-pregnene-21-ol- 3,11,20-trione are added and the reaction is permitted to proceed as above described.

The product is extracted with chloroform and isolated by evaporation to dryness. Recrystallization of the residue yields crystalline 1,4-pregnadiene-21-ol-3,11,20-trione.

As will be evident from the foregoing, the carbon side chain remains undisturbed in my process, i.e., it is not split off in whole or in part, Whether it contains but a single carbon atom, as in 17-methyl-testosterone, or contains two carbon atoms, saturated or unsaturated, as in pregnane compounds and in ethinyl testosterone, or contains more complex side chains, as in the sapogenins and pseudo-sapogenins, and as my experiments have indicated, in other steroids, like the bile and cholanic acids and their norand bisnor-derivatives.

With reference to the acetylation of the dienes described in Examples 1 and 2, it will be evident that other esters of the diene derivatives can be similarly prepared by reaction with the anhydride of the acid or with its chloride in known manner or by utilizing any of the known methods for preparing esters. These esters, especially the 2l-esters, as a class are within the presence of my invention and I have heretofore called such esters pharmaceutically acceptable esters by which term I mean those esters commonly used in steroid hormone therapy. Representative of such esters those formed from hydrocarbon carboxylic acids and dicarboxylic acids.

having up to 10 carbon atoms. While the acetates are most commonly used, other lower alkanoyl esters of the various hydroxylated dienes may be produced, such as the formates, propionates, butyrates, and valerates, cyclopentyl propionates, and likewise the esters of other nontoxic acids, like the benzoates, and also the neutral and acid esters of polybasic acids, like succinic, maleic, malic, citric, tartaric, phthalic and hexahydrophthalic. In the case of the acid esters, the metal salts can be formed in the usual manner by reaction with the hydroxide, carbonate or bicarbonate of the metal, as of the alkali and alkaline earth metals. Esters of polybasic inorganic acids such as sulfates, phosphates, and the like are also preparable from the free 20-01 or as indicated heretofore such grouping may be present during the fermentation.

Instead of forming the 1,4-dienes of cortical hormones such as cortisone and hydrocortisone, and subsequently csterifying the products, the corresponding esters of the monoenes and of their intermediates can be subjected to the process of the present invention and will yield the dienes of the esters. However, as above indicated, by suitable control of the reaction conditions the 21-alcohols can also be obtained from the 2l-esters. Thus, in Example 1, cortisone can be replaced by its 21-ester, as in Example 4, or by its 17u,21-diester (such as the acetate ester), or by S-pregnen-B,17a,21-trio1-11,20dione ZI-acetate or 17a,21-diacetate, or 3,17u,21-triacetate or other esters; while in Example 2, hydrocortisone can be replaced by its 2l-acetate, r l7a,21-diacetate, or 11[-3,17a- 21-triacetate; or by -pregnen-3,11p,17a,21-tetrol-20-one 3,21-diacetate, 3,17ct,21-triacetate or 3,11/3,17a,2l-tetraacetate. The polyesters can in all cases be mixed esters, like 3-propionate 21-acetate.

As already stated, Where a 3-hydroxy intermediate is employed, the formation of the diene derivative will be accompanied by an oxidation of the hydroxyl group to a keto group with a shifting of the double bond to the 4,5-position; while in the case of a 3-ester, such oxidation step will be preceded by a hydrolysis of the 3-ester group.

In place of cortisone, there can be employed as starting material 5-pregnen-3,17a,21-triol-11,20-dione, 5preghen-3,17a,20,21-tetrol-11-one or their S-acctates or other esters which are non-toxic toward the microorganism or inhibiting toward its enzyme; while similarly, in place of hydrocortisone, there can be used 5-pregnen-3,11fl,17a, 21-tetrol-20-one, 5-pregnen-3,11,8,17ot,20,2l-pentol, or their S-acetates or other esters. As indicated above, the 20-hydroxy group will in each case be oxidized to a keto group.

The following are examples of pharmaceutical compositions prepared in accordance with the invention and containing as the active hormone substance a 1,4pregnadiene compound (preferably an anti-inflammatory compound) of my invention:

EXAMPLE A M g./ tab. l-dehydrocortisone 5.00 Lactose 83.85 Starch 9 .50 Gelatin 1. 19 Magnesium stearate 0.46

This mixture provides a tablet for oral use in the treatment of rheumatoid arthritis and kindred conditions. The quantity of l-dhydrocortisone may be increased to about 30 mg. per tablet, but for tri-daily administration, 5 to mg. for each administration is usually suificient as maintenance doses.

EXAMPLE B Mg./tab. l-dehydrocortisone 5.0 Acetophenetidin 162.0 Caffeine, anhydrous 32.0 Acetylalicylic acid 227.67 Gelatin 5.0 Corn starch 55.33 Stearic acid 3.0

EXAMPLE C Mg./ml. l-dehydrocortisone, microcrystalline, sterile 5.0

Potassium dihydrogen phosphate, C.P. (KH PO 6.0 Disodium hydrogen phosphate, C.P. anh.

(Na i-IP0 12.0 Polysorbate 80, U.S.P. (Tween -Atlas) 0.4 Sorbitan monolaurate (Span 20Atlas) 0.4 Thimerosal, N.F. (Merthiolate--Lilly) 0.1

Water for injection, U.S.P., to make 1.00 ml.

This aqueous suspension is suitable for subcutaneous and intramuscular injection, and also as an ophthalmic preparation.

EXAMPLE D l-dehydrocortisone, microcrystals, 0.5% in a vehicle containing:

Percent Sodium chloride 0.9

Sodium carboxymethylcellulose 0.4 Polyoxyethylenesorbitan mono-oleate 0.4 Benzyl alcohol 0.9

Water for injection, q.s.

This mixture provides a suspension for intra-articular injection at the site of inflammation.

EXAMPLE E l-dehydrocortisone mg 5.0 Dimethylacetamine, q.s. ml 1.0

This infusion is to be diluted with 500 ml. 50% dextrose injection U.S.P. before administration by intravenous injection.

EXAMPLE F Mg./ml. l-dehydrocortisone, microcrystalline 5.0 Chloroprophenpyridamine gluconate, Schering 3.0 D-sorbitol 43.0

Benzalkonium chloride, U.S.P. 0.25 Distilled water, to make 1.0 ml.

The above mixture can be applied as a nasal spray.

EXAMPLE G Mg./ml. l-dehydrocortisone, microcrystalline 5.0 Aluminum monostearate 20.0 Propylparaben, U.S.P. 1.0 Sesame oil, U.S.P., to make 1.0 ml.

This oil suspension can be injected intramuscularly or subcutaneously.

EXAMPLE H Mg./g. l-dehydrocortisone, microcrystalline 5.0 Methylparaben, U.S.P. 1.7 Propylparaben, U.S.P. 0.3 Liquid petrolatum, U.S.P. 120.0 Anhydrous lanolin (wool fat), U.S.P. 200.0 White petrolatum, U.S.P 673.0

This mixture forms as ointment suitable for ophthalmic uses.

The above provides an antibacterial hormonal ointment suitable for ophthalmic treatment.

2Z1 EXAMPLE J Mg./g. l-dehydrocortisone, microcrystalline, sterile 5.0

Sodium sulfacetamide, anhydrous (Schering) 100.0

Methylparaben (U.S.P. XIV) 0.50 Propylparaben (U.S.P. XIV) 0.10 Arlacel 20 (Atlas) 1.00

Mineral oil (U.S.P. XIV) Kaydol 250.0 White petrolaturn (U.S.P. XIV), Protopet IS, to

make 1000.0 mg.

This ointment composition can be used for ophthalmic purposes.

EXAMPLE K Mg./ml. l-dehydrocortisone, microcrystalline 5.0 Chloroprophenpyndamine maleate, Schering 2.50

Sodium dihydrogen phosphate, monohydrate, re-

agent 4.603 Disodium hydrogen phosphate, anhydrous, re-

agent Sodium chloride, reagent 4.30 Benzalkonium chloride, U.S.P. 0.25 Distilled water, U.S.P., to make 1.00 ml.

This composition provides an ophthalmic suspension.

EXAMPLE L Mg./ml. l-dehydrocortisone, microcrystalline 5.0. Sodium sulfacetamide (monohydrate) 100.0.

Sodium dihydrogen phosphate, A.R., monohydrate 1.25 (1.087 anhydrous). Disodium hydrogen phosphate,

A.R., anhydrous 0.50. Sodium thiosulfate, U.S.P., re-

agent pentahydrate 1.57 (1.00 anhydrous). Methylparaben, U.S.P. 0.50. Propylparaben, U.S.P. 0.10.

Distilled Water (sterile), to make 1.00 ml.

This composition provides an ophthalmic suspension containing a sulfa drug as an antibacterial agent.

Distilled water (sterile), to make 1.00 ml.

This mixture is also an ophthalmic suspension with antibacterial properties.

EXAMPLE N Mg./ml. l-dehydrocortisone, microcrystalline, sterile 5.0 Sodium chloride, C.P 4.300 Disodium phosphate, C.P., anhydrous (Na HPO 4.730

Monosodium phosphate, C.P., monohydrate (Nal-I PO H O) 4.603 Benzalkonium chloride, U.S.P 0.200

Distilled water, U.S.P., to make 1.000 ml.

This composition is useful as an ophthalmic suspension.

no arm: EXAMPLE 0 Mg./g. l-dehydrocortisone 5.0 Zinc stearate, U.S.P 60.9 Polyethylene glycol 6000 (Carbowax 60-00) 122.0 Polyethylene glycol 1500 (Carbowax 1500) 421.3 Propylene glycol, U.S.P 345.2 Distilled water 45.6

This mixture forms a creamy composition for topical use.

EXAMPLE P Mg./g. l-dehydrocortisone 5.0 Neomycin sulfate 5.0 Zinc stearate, U.S.P 60.0 Polyethylene. glycol 6000 (Carbowax 6000) 122.0 Polyethylene glycol 1500 (Carbowax 1500) 421.3 Propylene glycol, U.S.P 345.2 Distilled water 40.6

This is likewise a cream for topical use, but is also antibacterial in character.

EXANIE'LE Q Mg./g. l-dehydrocortisone 5.0 Tetracycline hydrochloride 30.0 Neomycin sulfate 5.0 Zinc stearate, U.S.P 60.9 Polyethylene glycol 6000 (Carbowax 6000) 122.0 Polyethylene glycol 1500 (Carbowax 1500) 421.3 Propylene glycol, U.S.P 345.2 Distilled Water 40.6

The above mixture yields a cream suitable for topical application.

In the above formulations, l-dehydrocortisol (l-dehydrohydrocortisone) may replace the l-dehydrocortisone or mixtures of these two compounds may be employed in any proportions. The corresponding 9u-fluoro or chloro-compounds may be used in similar pharmaceutical preparations, and they may be mixed in any suitable proportion With l-dehydrocortisone or l-dehydrocortisol. The n -compounds may also be employed in admixture V with cortisone or hydrocortisone in difierent proportions.

Thus, in the tablets, emulsions, suspensions, infusions and creams hereinabove described, up to about 75% of the 1- dehydrocortisone can be replaced by an approximately equal weight of l-dehydrocortisol and a minor proportion of n -derivatives (say, up to about 40% can be replaced by cortisone or hydrocortisone, or a mixture of the two known compounds.

It will be understood that in the various pharmaceutical compositions prepared in accordance with the invention, the concentration of the l-dehydrocortisone or l-dehydrocortisol or of their 9a-halogeno derivatives will be such that an amount of the composition which can be conveniently administered during each treatment will contain suflicient quantity of such 1,4-pregnadiene which will elicit a proper therapeutic response. In the case of l-dehydrocortisone and l-dehydrocortisol this will be a dosage unit containing about 5 to 30 g. However, such an amount may be less for more potent ingredients. Thus, in the case of an infusion, the amount of solution which can be conveniently and safely injected during each administration will contain 5 to 30 mg. of the l-dehydrocortisone or l-dehydrocortisol. Similarly, in the case of a tablet, the concentration of the A -compound or compounds will be such that a tablet (by which term is included pills, capsules and other unit dosage forms) of a size which can be conveniently swallowed, will contain a therapeutically effective dose.

I claim:

1. The M-pregnene having a keto group at the 3- and 20-positi0ns, hydrogen at the 9a-pOSitiOn, hydroxy at the t-POS11Z101'1, and a member of the group consisting of hydroxy and pharmaceutically acceptable esters thereof at the 2l-position, and being characterized by the presence of a double bond in the 1-position and a member of the group consisting of keto and fl-hydroxy at the ll-position.

2. 1,4-pregnadiene-17u,21-diol3,11,20trione having a melting point of about 230-232 C. with decomposition and a specific rotation of about +175 in dioxane.

3. A 21-pharmaceutically acceptable ester of 1,4-pregnadiene-17a,21-diol-3 ,11,20-trione.

4. 1,4 pregnadiene 17a,21-diO1-3,1 1,20-trione 21-acetate.

5. IA-Pregnadiene-I1 3,170;,21-triol-3,2l-dione having} a melting point of about 239241 C. with decomposition and a specific rotation of about +107 in dioxane.

6. A pharmaceutically acceptable ester of 1,4-pregnadiene-l1 3,17a,21-triol-3,2O-dione.

7. 1,4 pregnadiene-l1p,17a,21-triol-3,20-dione 21-acetate.

8. A pharmaceutical preparation comprising an effective amount of an anti-inflammatory composition of matter in the form of a A -pregnene having a keto group at the 3 and -positions, hydrogen at the Set-position, hydroXy at the 17oc-pOSilZlOI1 and a member of the group consisting of hydroxy and pharmaceutically acceptable esters thereof at the 21-position, and being characterized by the presence of a double bond in the 1-position and a member of the group consisting of keto and B-hydroxy at the ll-position, mixed with a non-toxic pharmaceutical carrier.

9. The process for the application of anti-inflammatory therapy which comprises administering an effective amount of a pharmaceutical formulation comprising a composition of matter in the form of a M-pregnene having a keto group at the 3- and 20-positions, hydrogen at the 9aposition, hydroxy at the 17a-position and a member of the group consisting of hydroxy and pharmaceutically acceptable esters thereof at the 21-position, and being characterized by the presence of a double bond in the l-position and a member of the group consisting of keto and B-hydroxy at the ll-position, mixed with a non-toxic pharmaceutical carrier.

10. The process for the application of anti-inflammatory therapy which comprises administering an effective amount of a pharmaceutical formulation comprising a composition of matter selected from the group consisting of 1,4- pregnadiene-17a,21-diol-3,11,20-trione and the 21-pharma ceutically acceptable esters thereof.

11. The process for the application of anti-inflammatory therapy which comprises administering an eifective amount of a pharmaceutical formulation comprising a composition of matter selected from the group consisting of 1,4- pregnadiene-l1,8,17u-21-triol-3,20-dione and the 21-pharmaceutically acceptable esters thereof.

12. The A -pregnene having a keto group at the 3- and 20-positions, hydrogen at each of the 9a and 17a-positions and a member of the group consisting of hydrogen,

hydroxy and pharmaceutically acceptable esters thereof at the 21-position, and being characterized by the presence of a double bond in the 1-position and a member of the group consisting of (H, H), O and (H, OH) at the 11- position.

13. 1,4-pregnadiene-3,ZO-dione.

14. 1,4-pregnadiene-1118,21-diol-3,20-dione.

15. 1,4-pregnadiene-21-0l-3,1 1,20-trione.

16. 1,4-pregnadiene-21-ol-3,20-dione.

17. 1,4-pregnadiene-11,6,21-dio1-3,20-dione 21 lower alkanoate.

18. 1,4-pregnacliene-21-ol-3,11,20-trione 21-lower alkanoate.

19. 1,4-pregnadiene-21-ol-3,20-dione 21 lower alkanoate.

20. 17a-ethinyl-1,4-androstadiene-17/3-ol-3-one.

21. 22a-25D-spirosta-1,4-diene-3-one.

22. 1,4-pregnadiene-l7u,20,21-triol-3-one.

23. A 21-lower alkanoate of 1,4-pregnadiene-17a,20,21- triol-3-one.

24. A A -pregnadiene-11fi,l7oc,21-triol-3,20 dione of the formula CHzOR CHzOR wherein R is a member selected from the group consisting of hydrogen and lower alkanoyl.

References Cited in the file of this patent UNITED STATES PATENTS 2,774,775 Korman et al Dec. 18, 1956 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3, 134, 718 May 26, 1964 Arthur Nobile Column 2, 1ine'2l,for "-pregnane" read -pregnene line 43, for "modifications" read modification column 3, line 39, for "115 17a,2" read 115 l7 ,21 same column 3, lines 62, 63, 64 and 65 should appear as shown below instead of as in the patent:

Y=CO-CH OR (R=H or acyl) when .OH or. .H Y=OH when 2 .CECH or .CH or. 0H

lines 66 to 75, the right-hand formula should appear as s hown below instead of as in the patent: CH3

column 4, line 55, for "transformation" read transformations column 6, lines 1 to 10, the right-hand portion of formula (5) should appear as shown below instead of as in the patent:

same column 6, lines 26 to 35, f

ormula (7) should appearas shown below ins tead of as in the patent:

HO fi same as (5) same column 6, lines 51 to 75, formula (9) sh shown below instead of a s in the patent: CH OOCCH ould appear as CH2OH =0 CH OOCCE-i column 7 TABLE I, under the heading "Products", lines 37 and 38 thereof, for "l 4Pregnaiene3, 20-dione. read 1 ,4- Pregnadiene3, 20-dione. same column '7 line 39 for "hermone" read hormone column 8, lines 15 and 16, for "l-dehydrocortisone" read l-Dehydrocorti sone column 9 line 50 for "diones" read dienes column 10, line 50, for "adminsitered" read administered line 64, for

"01;" read to column 13, line 69, for "steriod" read steroid column 15, line 43, for "asptically read aseptically line 65, after "ml strike out "of"; column 16, line 32, for "stream" read steam column 1'? line 18, for "in", first occurrence, read an column 18, line 4 after "reaction" strike out "of" column 19, line 6, for "20-01" read 2l-0l line 62, for "l--dhydrocortisone" read 1-dehydrocortisone column 20, line 30, for "Dimethylacetamine" read Dimethylacetamide Signed and sealed this 22nd day of December 1964.

(SEAL) Attest:

ERNEST W. SWIDER EDWARD J BRENNER Attesting Officer Commissioner of Patents 

1. THE $4-PREGENENE HAVING A KETO GROUP AT THE 3- AND 20-POSITIONS, HYDROGEN AT THE 9A-POSITION, HYDROXY AT THE 17 A-POSITION, AND A MEMBER OF THE GROUP CONSISTING OF HYDROXY AND PHARMACEUTICALLY ACCEPTABLE ESTERS THEREOF AT THE 21-POSITION, AND BEING CHARACTERIZED BY THE PRESENCE OF A DOUBLE BOND IN THE 1-POSITION AND A MEMBER OF THE GROUP CONSISTING OF KETO AND B-HYDROXY AT THE 11-POSITION.
 8. A PHARMACEUTICAL PREPARATION COMPRISING AN EFFECTIVE AMOUNT OF AN ANTI-INFLAMMATORY COMPOSITION OF MATTER IN THE FORM OF A $4-PREGNENE HAVING A KETO GROUP AT THE 3- AND 20-POSITIONS, HYDROGEN AT THE 9 A-POSITION, HYDROXY AT THE 17 A-POSITION AND A MEMBER OF THE GROUP CONSISTING OF HYDROXY AND PHARMACEUTICALLY ACCEPTABLE ESTERS THEREOF AT THE 21-POSITION, AND BEING CHARACTERIZED BY THE PRESENCE OF A DOUBLE BOND IN THE 1-POSITION AND A MEMBER OF THE GROUP CONSISTING OF KETO AND B-HYDROXY AT THE 11-POSITION, MIXED WITH A NON-TOXIC PHARMACEUTICAL CARRIER. 